| Description | Products contentS665683Component1mL5mLStorageS665683A2×Super Kfx MasterMix1 mL5×1 mL -20℃. Avoid freeze/thaw cycle.S665683BddH2O1 mL5×1 mL -20℃. Avoid freeze/thaw cycle.Products IntroductionThis product is a premixed system consisting of Super Kfx DNA Polymerase, Mg2+, Products contentS665683Component1mL5mLStorageS665683A2×Super Kfx MasterMix1 mL5×1 mL -20℃. Avoid freeze/thaw cycle.S665683BddH2O1 mL5×1 mL -20℃. Avoid freeze/thaw cycle.Products IntroductionThis product is a premixed system consisting of Super Kfx DNA Polymerase, Mg2+, dNTPs, PCR stabilizers and enhancers at a concentration of 2×. Super Kfx DNA Polymerase is a fast, high-fidelity DNA polymerase with high amplification efficiency, which possesses 5′-3′DNA polymerase activity and 3′-5′ exonuclease activity. With 5′-3′ DNA polymerase activity and 3′-5′ exonuclease activity, the enzyme has the advantages of strong amplification ability, high fidelity and high specificity, etc. 2×Mix has added unique amplification enhancement factors and extension factors, and the unique formula makes the whole reaction system very stable and easy to operate, which is suitable for the amplification of various fragments and templates. The product is suitable for gene cloning, second generation library amplification, targeted gene mutation, SNP amplification experiments. quality controlNo exogenous nuclease activity, can effectively amplify various templates; stored at 2-8℃ for one month, no obvious activity change.UsageThe following examples are conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and target fragment size.1. PCR reaction system All operations should be carried out on ice, please mix the components thoroughly after thawing, after use, please promptly return to -20 ℃ for storage. 2. PCR reaction system take note of1）Priority is given to three-step amplification; if the three-step method fails to amplify the target product or if the primer Tm value is greater than 68°C, try the two-step method.2）Denaturation: pre-denaturation of simple templates 98°C, 30s-1min, for complex templates, the pre-denaturation time can be extended to 3min. 3）Annealing: In general, the annealing temperature is 3-5℃ lower than the Tm value of the primers. If the desired amplification efficiency cannot be obtained, the annealing temperature should be changed in a gradient to optimize the results; if a non-specific reaction occurs, the annealing temperature should be increased appropriately.4）Extension: The extension time should be set according to the length of the amplified fragments and the complexity of the template, the amplification efficiency of this product is 4-6kb/min, for long fragments and templates with high complexity it is recommended that 2-4kb/min. 5）Cycling times: the number of cycles can be set according to the downstream application of the amplified product. If the number of cycles is too small, the amplification will be insufficient, and if the number of cycles is too large, the chance of mismatch will be increased, so the number of cycles should be minimized under the premise of guaranteeing the yield of the product... Read More | Inquire | Inquire | Inquire | Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed Reverse transcriptases are enzymes encoded in retroviruses viral genome. The enzyme is responsible for transcription of the viral RNA to produce a dsDNA that can be inserted into the host genome.Reverse transcriptases are multifunctional enzymes. These enzymes exhibit an RNA and DNA directed polymerase activity. In addition reverse transcriptases catalyze the degradation of RNA in an RNA-DNA hybrid. The exonucleolytic activity proceeds in a 5' ---> 3' direction. The RNA or DNA directed activity requires a template (RNA or DNA) and a primer. The following is a schematic illustration of the reaction:Unit definition: One unit incorporates 1 nanomole of tritiated dTMP into acid insoluble productsusing poly(A)•oligo(dT) 12-18 as the template-primer in 20 minutes at 37° C.ApplicationsHIV reverse transcriptase is used for research on the AIDS primer. However it can be substituted for AMV reverse transcriptase, which is mainly used to transcribe mRNA into double stranded cDNA, that can be inserted into prokaryotic vectors. The enzyme can also be used with either single stranded DNA or RNA templates to make probes for use in hybridization experiments. It can be used for labeling the termini of DNA fragments with protruding 5' termini. The enzyme can also be used to sequence DNAs by the dideoxy chain termination method of Sanger when the Klenow fragment of E. coli DNA polymerase I, or the T7 DNA polymerase yield unsatisfactory results.Reagents0.05 M Tris, pH 8.3, containing 0.008 M MgCl21 mg/ml polyadenylic acid in water (poly A)DNA primer:Oligo d(T)12-181 µ mole dTTP/mL stock solution[methyl-3H]-Thymidine 5'-triphosphate (3H-dTTP)dTTP-3H-dTTP working mix: Add 1-2 µL 3H-dTTP per mL of 100 nmol/mL dTTP in order to obtain 1 to 1.5 x 105 cpm/mL1% bovine serum albumin10% perchloric acid1% perchloric acidBuffer substrate reaction mixture: Prepare fresh, immediately before use:For each 1mL of reaction mixture required mix:0.7 mL Tris/HCl, pH 8.3, 0.008M MgCl20.3 mL 1 mg/mL poly(A) RNA template0.005 mL 0.02 mg/mL oligo d(T)12-18 DNA primer0.02mL 1% BSAEnzymedilute as needed wtih 0.05M Tris/HCl, pH 8.3, 0.008M MgCl2 containing 0.1 mg/mL (1%) BSAProcedurePipette into each tube as follows:Buffer substrate mix：0.1 mLdTTP-3H3-dTTP：0.1 mLEnzyme：5-10 µLIncubate 20 minutes at 37° C. Stop reaction by adding 1 ml 10% cold perchloric acid. Filter through 0.2µ manifold filters used with Millipore vacuum manifold. Wash four times using 2mL 1% cold perchloric acid/wash. Transfer filter to scintillation vials. Add 2mL Cellosolve (or 2-methoxyethanol) to dissolve filter. Filters become opaque upon addition of Cellosolve. Make sure filters are dissolved before proceeding. Add 10mL scintillation cocktail and count.Calculation... Read More |